THE STUDY OF THE PRESERVATIVE EFFECT OF A BLEND OF Tamarindus indica and Ziziphus abyssinica EXTRACTS IN EXTENDING THE SHELF- LIFE OF MEAT BALLS
ABSTRACT
The study is aimed at finding out if combining the two herbs would result in a more effective preservative than when each herb is used independently.Preservation using natural products is associated with the presence of antioxidants,antimicrobial,antifungal or dehydrating properties.
Fresh meatballs are made from meats that have not been previously cured,they are made from ground meat,either pork,beef or veal.
Meatballs are however susceptible to microbial spoilage and auto-oxidation. The artificial chemical preservatives used in meatball preservation.eg sodium metabisulphite may be replaced by natural extracts such as Tamarind and Ziziphus extracts which have both antimicrobial and anti-oxidant properties.
The extracts will be obtained from Tamarind andZiziphusseeds.The seeds will be dried and ground into powder followed by liquid/liquid extraction. The extraction was carried out by 400ml ethyl acetate and water for 100grams from each sample.
The fresh meatballs will be processed in the JKUAT meat workshop using the correct recipe. Fresh meatballs batch with no preservative will act as the control. Another batch with sodium metabisulphite and finally with the seed extracts.
The processed fresh meatballs will be stored at 40c and at 250c and the samples analyzed within a staggered period of 4 weeks for auto-oxidation,microbial load and colourchanges.The obtained results will be recorded, analyzed and presented appropriately by use of correct scientific statistical methods e.g. graphs.

1.0 INTRODUCTION
The consumption of ready-to-eat meals has grown, mainly because modern leaving allows little time to prepare meals. Despite the sophisticated technologies used in the production of this type of food, lipid oxidation remains the most important mechanisms of quality deterioration in meat-based products, such as meat balls and sausages which are frequently introduced in some ready-to-eat meals together with sauce.
Food lipids are principally triacylglycerides, phospholipids and sterols found naturally in most biological materials consumed as food and added as functional ingredients in many processed foods. As nutrients, lipids, especially triglycerides, are a concentrated caloric source, provide essential fatty acids and are a solvent and absorption vehicle for fat-soluble vitamins and other nutrients. The presence of fat significantly enhances the organoleptic perception of foods, which .,partly explains the strong preference and market advantage of fat-rich foods. As a class, lipids contribute many desirable qualities to foods, including attributes of texture, structure, mouth feel, flavor and color. However, lipids are also one of the most chemically unstable food components and will readily undergo free-radical chain reactions that not only deteriorate the lipids but also: (a) produce oxidative fragments, some of which are volatile and are perceived as the off-flavors of rancidity, (b) degrade proteins, vitamins and pigments and (c) cross-link lipids and other macromolecules into non-nutritive polymers. Free-radical chain reactions are thermodynamically favorable, and as a result, evolutionary selection has strongly influenced the chemistry, metabolism and structure of biological cells to prevent these reactions kinetically. However, the loss of native structure and the death of cells can dramatically accelerate the deteriorative reactions of lipid oxidation. The effects of all processing steps, including raw product selection, harvesting, storage, refining, manufacturing and distribution, on the quality of lipids in the final commodity are considerable. Certain key variables now known to influence oxidative processes can be targeted to increase food lipid stability during and after processing. Retention of or addition of exogenous antioxidants is a well-known consideration, but the presence and activity of catalysts, the integrity of tissues and cells, the quantity of polyunsaturated lipids and the structural properties of the final food product, including total surface area of lipids, and the nature of surfactant materials all play important roles in final product stability.
The oxidative degradation of unsaturated fatty acids produces volatile compounds, which contribute to the deterioration potentially toxic compounds such as malonaldehyde and oxysterols.(Junoszka,2010;selani et al,2011)
The use of refrigerated storage, modified atmosphere or vacuum packaging contributes to delaying lipid oxidation and to maintaining microbiological safety and quality parameters, such as color, odor, texture and even the nutritional value. A common practice to inhibit lipid oxidation in meat and meat products is to use synthetic antioxidants. However, growing concern among consumers concerning such chemical additives has led to search for natural additives, especially those of a plant origin, such as green tea and grape, Tarmarind,Ziziphusetc not only for their antioxidant activity but also for their antimicrobial properties.
Phenolic compounds are considered safe than synthetic antioxidants. Phenolic extracts prepared from plant sources are known to have antimicrobial effectives, although even if they are used it is not possible to completely eliminate the use of chemical antimicrobials.(Banon et al..2007;Garrido et al,2011,Liu et al,2010)
In the last few years, a number of laboratory studies have revealed the efficacy of plant extracts and phytochemicals as antimicrobials. These properties are attributed to the presence of secondary metabolites such as phenolics in essential oils and tannins in herbal extracts. Some examples of classes of natural materials that afford antimicrobial protection include essential oils such as tea tree oil, rosemary oil and turmeric oil; plant extracts such as rosemary extract, sage extract, lemon balm extract, green tea extract, Kaempferiagalanga extract, Neem leaf extract and oil, and isolated phytochemicals such as cinnamates, benzoates, eugenol.
In the meat industry, Sodium metabisulphite is the main synthetic antioxidant used since if inhibits peroxidation.(Mitsumoto et al 1991;Sahoo and Anjaneyulu 1997)

1.1 PROBLEM STATEMENT Concerns have been raised about cytotoxic, mutagenic and carcinogenic effects artificial preservatives such as sodium metabisulphite.Studies have shown opc(oligomericproanthocyanidins) to be more powerful antioxidants than Vit C,E and beta-carotene.-it contains flavonoids, phytochemicals that have powerful antioxidant effects.Under-utilization of Tarmarindusindica and Ziziphusabyssinica
1.2 JUSTIFICATION

Synthetic antioxidants tend to promote peroxidation reactions when consumed over a long period of time. They have been associated with diseases such as cancer. Natural antioxidants on the other hand do not have such negative effects hence need to find ways of obtaining and using natural antioxidants5.(Brown and Jessup,1999;Zieden et al;1999)
Using “natural green” plant extracts or their derived products in various food and beverage applications is an increasing trend in the food industry. Selection of these plant extracts and their application depends on their functional properties, availability, cost effectiveness, consumer awareness and their 456effect on the sensory attributes of the final product.Tarmarind and Ziziphus extracts are derived from their respective seeds that is extracted,dried and purified to produce polyphenolic compounds-rich extract that also has well documented antioxidant, antimicrobial and anti-inflammatory properties. This plant extract (polyphenol and proanthocyanidin rich compound) has potential antioxidant properties by inhibiting the lipid oxidation and warmed over flavors and antimicrobial activities against major food borne pathogens like Listeria monocytogenes, SalmonellaTyphimurium, Escherichia coli O157:H7, and Campylobacter jejuni in preventing pathogen contamination. Furthermore, they have demonstrated synergism in antimicrobial activity when used in combination with organic acids (malic, tartaric acid, benzoic acids etc.), bacteriocins like nisin or chelating agents like EDTA in various model systems including fresh produce (fruits and vegetables), raw and ready-to-eat meat products and poultry.
Further more,Tarmarind and Ziziphus have been under-utilized,hence by this,they can be better utilized.
1.3 OBJECTIVES
1.3.1 MAIN OBJECTIVES
To determine the effectiveness of Tamarind and Ziziphus extracts versus sodium metabisulphite on the shelf life extension of meatballs.
1.3.2 SPECIFIC OBJECTIVES * To prepare Tarmarind and Ziziphus extracts using Liquid/liquid extraction method by methanol and water. * Preparation of the meatballs in the Jkuat meat workshop. * Treatment of the samples with different concentrations of the extract and a control using sodium metabisulphite. * Keeping the samples at refrigeration temperatures of 40c and carry out microbial analysis,rancidity level evaluation and sensory evaluation of the products.
1.4 HYPOTHESSIS FOR THE RESEARCH
The use of naturally occurring preservative extracts such asTamarindusindica andZiziphusabyssinica will lengthen the storage time of the meat products.

2.0 LITERATURE REVIEW
Tamarind scientifically known as Tamarindus indica derives from the Persian word Tamar e hind means ‘Indian date’ is a tree in the Fabaceae family. The genus Tamarindus is having an single species only.
The flowers of the tamarind tree are very ordinary with nice spreading branches and a canopy of bulging flora. The tree is much admired as an avenue, park or garden tree as it has very useful fruits and the timber of this tree is highly prized. It has a short but strong trunk to bear the weight of its wide and extensive top. The almost black bark is thick and some longitudinal and horizontal cracks cover it well. The tree can achieve the height of 27 meters.
The fruit also called as the pod is about 12 to 15 cm in length with a hard brown shell. The fruit has fleshy, juicy and acidulous pulp. When matured it is colored brown or reddish brown. The tamarinds grown in Asia have longer pods containing about 6 to 12 seeds whereas in Africa and West Indies the varieties are short pods containing only about 1 to 6 seeds. The seeds too are somewhat flattened and glossy brown. A tamarind is excellent when it is sweet and sour in taste and high in acid, sugar, vitamin B and interestingly for a fruit, calcium.
The fruit of tamarind tree has numerous usages. The pulp is used as an important ingredient in the curries. There are some commercial uses too. It is preserved and also sold in the markets. It is also used as a laxative in medicine. People make powder from grinding the seeds and boil it to paste with gum and make strong cement. A substitute for wheat or other flour can also be obtained from them that are used by the people. The stalks of the seeds have been employed for road surfacing as well. The scientists also discovered that the seeds could make a cheap but efficient substitute for cereal starch that is used for making the cotton yarn in proper size, for jute fabrics and for woolens. Further, the leaves and flowers of the tree are also quite useful. An infusion from the leaves can make a fine yellow dye that is used to give a green color to silks. Though hard and very difficult to work on, the timber of the tree is of high value. People widely use this wood for making wheels, mallets, furniture, oil and sugar mills, etc.
Tamarind juice is a mild laxative, is used to treat bile disorders, lowers cholesterol, and promotes a healthy heart. Tamarind is use as a gargle for sore throats, and as a drink to bring relief from sunstroke. It is used as a diuretic remedy for bilious disorders, jaundice and catarrh. It is a good source of antioxidants that fight against cancer. Tamarind helps the body digest food. Tamarind has a variety of uses. The unripe fruit is acid in taste, whereas the pulp of the ripe fruit is both sweet and acid and is cooling, carminative, digestive and laxative. It is anti-bilious and anti-scorbutic.
The jujube is a member of the buckthorn family, or Rhamnaceae. Its botanical name is Ziziphuszizyphus (formerly Zizyphusjujuba ), and its common names are common jujube, Chinese date, and Chinese jujube. Though the plant's origin is probably Syria, it was distributed throughout much of the Mediterranean region at least 3,000 years ago and today is most widely grown in China. The tree can reach a height of 5-12 m, with shiny-green leaves, and sometimes thorns. The many inconspicuous flowers are small, greenish or white, and produce an olive-sized fruit that is a drupe. Jujube has been a part of Chinese medicine for at least 2,500 years and is mentioned in the Classic of Odes, a 6th century BCE anthology of Chinese poetry. The fruit has a pleasant taste and high nutritional value, and is often used to disguise unpalatable prescriptions. The fruit is a drupe, varying from round to elongate and from cherry-size to plum-size, depending on the cultivar. It has a thin, edible dark red skin surrounding whitish flesh of sweet, agreeable flavor. The single hard stone contains two seeds. Confusingly, Jujube is also the name of a tiny fruit-flavored candy with a hard, gelatinous texture, but the name is the only connection between the two foods. It thrives best in warm, dry climates; but will withstand winter temperatures down to -20 F. Fruit is generally dark brown when ripe, oval to pyriform in shape, 1 to 2 inches diameter, with a single stone. Fruit will dry if left on tree, similar to figs. Skin is smooth and thin until drying of fruit occurs, then becomes wrinkled. Pulp is dryer than in most fruits.
Food: The sweet fruits are edible, and the leaves may be cooked as a vegetable.
Fodder: Jujube is browsed by livestock in spite of its thorns, and in Democratic Republic of Congo, it is cultivated as a fodder crop.
Apiculture: Z. abyssinica is an excellent tree for bees, both pollen and nectar being easily available.
Fuel: The species is a source of firewood and is used in the production of charcoal.
Timber: The dark brown to black wood is heavy, hard and resistant to termites and borers. It is used mainly as poles to fence kraals and villages and to cover graves. It is also used for furniture, interior work and carving.
Tannin or dyestuff: The bark yields a cinnamon-coloured dye.
Medicine: Ash from the burnt leaves is mixed with salt and applied on the throat to relieve tonsillitis. A fomentation of steaming hot leaves soaked in boiling water are used as on the chest to treat pneumonia.

3.0 METHODOLOGY Tarmarind SAMPLE,100g
Tarmarind SAMPLE,100g
Ziziphus sample,100kg
Ziziphus sample,100kg
FLOW CHART;

EXTRACTION WITH METHANOL AND WATER

EXTRACTION WITH METHANOL AND WATER

FRYING FOR SENSORY EVALUATION
FRYING FOR SENSORY EVALUATION
COLD STORAGE FOR LAB ANALYSIS
COLD STORAGE FOR LAB ANALYSIS INGREDIENT IN FRESH meatball MAKING INGREDIENT IN FRESH meatball MAKING

meatball MAKING meatball MAKING

SODIUM METABISULPHITE
SODIUM METABISULPHITE BLEND OF THE EXTRACTS BLEND OF THE EXTRACTS

MICROBIAL ANALYSIS STAGGERED IN FOUR WEEKS
MICROBIAL ANALYSIS STAGGERED IN FOUR WEEKS

RANCIDITY TESTS STAGGERED IN FOUR WEEEKS
RANCIDITY TESTS STAGGERED IN FOUR WEEEKS

DETERMINATION OF SENSORY CHANGES
DETERMINATION OF SENSORY CHANGES

RESULTS ANALYSIS RESULTS ANALYSIS

3.1 ACQUISITION OF RAW MATERIALS AND SAMPLES
Meat will be bought from a butchery in Juja and used to make meat balls
Tamarindusindica and Ziziphusabyssinica were obtained from Kapenguria in West Pokot.
3.2EXTRACTION PROCEDURE
3.2.1 Preparation of Extract
45g portion of the herb Tamarind was extracted using distilled water to simulate the traditional practice by the pastoralists (Bautista-Banos et al., 2003). The water mixture was boiled for one hour and then left to cool for one day. The mixture was later centrifuged at 20,000rpm for 10 minutes at a temperature of 4°C using a Kokusan Centrifuge from Kokusan Corporation (Model 2000C, Tokyo Japan). The supernatant was filtered using No. 1 Whatman filter paper and the filtrate evaporated to dryness at about 80±2°C.
A 45g portion of Ziziphus was extracted using the cold method with methanol as the solvent. In cold extraction, the herb was immersed in the extracting solvent and placed in opaque glass container. The container was shaken for 1 day to ensure sufficient contact using a KikaLabortechnik Shaker, (Model KS 250 Basic, Staufen, Germany). The mixtures was left to stand for four days in a dark enclosure at 25 ± 2°C and then filtered. The filtrate obtained from the product of cold extraction was evaporated to dryness under vacuum at 80 ± 2°C using a rotary evaporator(Model RE 100, Staffordshire, England). All the dried samples were put in labeled and tightly corked light proof glass container and store at 4 ± 2°C.
The two extracts MTI and AZA were mixed in the ratios provided below on weight to weight basis totaling 1gm and diluted to 100ml with distilled water

AZAgms | MTI | 1 | 1 | ¼ | ¾ | ¾ | ¼ | 1 | 0 | 0 | 1 |
Take each of the potions and test them for
1. Antioxidation capacity
3.2.2 Determination of the free radical scavenging activity (FRSA) of plant extracts
To measure the antioxidant capacity of the extracts the hydrogen donating or free radical scavenging activity, was measured using the stable radical DPPH. To extracts of various concentrations (0.02 – 0.1 mg/ml) 1ml methanol was added together with 0.5 ml of 1 mM DPPH solution in methanol. A blank solution was prepared containing 1 ml of methanol and 0.5 ml of 1mM DPPH in methanol. The experiments were carried out in triplicates. The test tubes were incubated for 15 min, methanol was used to zero the spectrophotometer and the absorbance was read at 517 nm. The radical scavenging activity was calculated using the following formula:
% inhibition of DPPH = {(AB – AA)/AB} x100
Where:
* AB is the absorption of blank sample and * AA is the absorption of tested extract solution.
The results are expressed as percentage inhibition of DPPH and mean inhibitory concentrations (IC50) determined from a plot of absorbance of DPPH versus concentration of extract.
4.0 RESULTS | AZAgms | MTI | CONC.Mg/ml | Absorbance517nm | A | 1 | 1 | 0.02 | 0.191 | B | ¼ | ¾ | 0.02 | 0.298 | C | ¾ | ¼ | 0.02 | 0.316 | D | 1 | 0 | 0.02 | 0.343 | E | 0 | 1 | 0.02 | 0.400 |

CALCULATIONS
% inhibition of DPPH = {(AB – AA)/AB} x100
Absorbance of Blank=0.487
A: % inhibition of DPPH={(0.487-0.191)/0.487} =60.8%
B: % inhibition of DPPH ={(0.487-0.298)/0.487} =38.8%
C: % inhibition of DPPH ={(0.487-0.216)/0.487} =55.6%
D: % inhibition of DPPH ={(0.487-0.343)/0.487} =29.57%
E: % inhibition of DPPH ={(0.487-0.400)/0.487}=17.9% | AZAgms | MTI | CONC.Mg/ml | Absorbance517nm | %inhibition of DPPH | A | 1 | 1 | 0.02 | 0.191 | 60.8 | B | ¼ | 3/4 | 0.02 | 0.298 | 38.8 | C | ¾ | 1/4 | 0.02 | 0.216 | 55.6 | D | 1 | 0 | 0.02 | 0.343 | 29.57 | E | 0 | 1 | 0.02 | 0.400 | 17.9 |

5.0 DISCUSSION AND CONCLUSION All aerobic organisms have antioxidant defense systems to offset harmful effects caused by free radicals. In the case of failure of the antioxidant defense system, antioxidants need to be supplemented from outside sources. Antioxidants can be found naturally in foods (Kedare and Singh, 2011). A majority of antioxidants naturally present in foods occur in phenolic structures and especially in flavonoid structures. In addition, antioxidants are added to nutrients to prevent deterioration in their taste, smell, and color. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG) can be included in this group, which are known as synthetic antioxidants (Koksal and Gulcin, 2008). The high cost of natural antioxidants has led to the use of synthetic antioxidants. However, studies conducted subsequently have demonstrated that synthetic antioxidants have toxic effects and, consequently, restrictions have been imposed on their use. Therefore, researchers have focused their studies on plant-derived natural antioxidants (Kulisic et al., 2004). Many data gathered in recent years indicate the participation of free radical processes in the emergence of such lifestyle diseases as atherosclerosis, heart attack, stroke, cancer, diabetes,senile cataracts and accelerated aging. The presence and distribution of numerous hydroxyl groups in the chemical structure of polyphenols make them excellent antioxidants. They are able to chelate transition metal ions, particularly those of iron and copper, which are involved in initiating free radical chain reactions. The antioxidant activity of the leaf extracts was assessed by spectrophotometry of the presence of the DPPH radical, which is often used to compare the activity of plant extracts. DPPH is a stable free radical which dissolves in methanol and shows characteristic absorption at 517 nm. When an antioxidant scavenges free radicals by hydrogen donation, the DPPH assay solution becomes lighter in color (Molyneux,2004; Villańo et al., 2007) DPPH free radical can easily receive an electron or hydrogen from antioxidant molecules to become a stable molecule. A solution of DPPH radicals prepared in methanol is converted into DPPH-H molecule in the presence of antioxidant agent: DPPH°+A-H DPPH-H+A° Discoloration occurs due to the decreasing quantity of DPPH radicals in the environment, reflected by a decrease in the absorbance of the sample extracts. The discoloration of the DPPH therefore reflects the radical scavenging activity of the extract. Hence the the greater the degree of discoloration, the lower the absorbance values, hence high levels of antioxidants. The antioxidant properties of phenolic compounds originate from their properties to proton loss, chelate formation and dismutation of radicals. The decreasing absorbance reflects a high antioxidant percentage. The most effective blend depicting a high antioxidant % is A & B

6.0 PENDING WORK Evaluation of antibacterial activity of herb extracts
Pure cultures of the test microorganisms will be inoculated into nutrient broth (Oxoid, England), incubated for 24 h at 37°C, diluted with sterile nutrient broth to a density of 9×108 cfu/ml by serial dilution. Sterile disposable plates will be used and appropriate media be prepared and poured into sterile disposable plates according to AOAC method 966.23 (AOAC, 1995). Inoculation of the prepared plates with the organism will be done using a sterilized pipette to transfer 0.1ml of the suspensions into the plates followed by spreading with a Canards rod to achieve uniform spread on the plate. MEAT BALL MAKING
The meat balls will be processed in the JKUAT meat workshop using the following recipe for 3kg meat; 1. Meat 3kg
Four groups of meat balls are to be prepared in duplicate in commercial conditions. The meat which will have been stored at -180c,will minced(5mm),at a ratio of 70% lean pork meat and 30% back fat.200-300mg sodium metabisulphate, seed extracts per kg was added. The meat balls were prepared then fried.

ANALYSIS OF THE SAMPLES
The samples will be analyzed using TBA assay(Thiobarbituric assay) to assess lipid oxidative stability.
Method
Lipid peroxidation: Malondialdehyde (MDA), the by-product of L.P.O forms adduct with TBA. On boiling, it produces pink colored complex, which absorbs maximally at 532 nm.
0.1 ml sample, 0.1 ml Tris-HCl buffer, 0.1 ml FeSO4 and 0.1 ml Ascorbic acid are to be added in a test tube then 0.6 ml DDW added to make the volume 1.0 ml. It will be incubated at 370C for 15 min. Then 1.0 ml TCA and 2 ml TBA will be added to the reaction mix. Tubes will be plugged and incubated for 15 min. at boiling water. Centrifugation will be done at 3000 rpm for 10 min. Readings will be taken at 532 nm.(Izunimoto et al1990)
Calculations:
The concentration of MDA is calculated using extinction coefficient of MDA-TBA complex which is 1.56 × 105 M-1 cm-1 and the results are expressed as n moles MDA/mg protein.

SENSORY EVALUATION/ANALYSIS
The sensory assessment of the fried meatballs subjected to different treatments will be undertaken by a group of forty untrained panelists from the Department of Food Science and Technology. Each of the panelists will be presented with a deep fried fresh meatball sample. One will be the control with no preservative, another with sodium metabisulphate as preservative and the last one with the seed extracts. The samples will be coded using random numbers and placed on plastic plates and presented to the panelists. Each panelist will be asked to evaluate the samples for color, appearance, flavour, texture, hardness and general acceptability. Evaluation will be done using a 9-point hedonic scale. The parameters evaluated will be all scored between 9 (like extremely) and 1 (Dislike extremely) using a questionnaire.

MICROBIOLOGY
The samples (10 g) used for microbiological analysis will be aseptically mixed with 90 ml peptone water in sterile plastic bags and blended using a stomacher). Aliquots will be serially diluted (1:10) in peptone water. Sample dilutions (1 ml) will be plated and incubated following standard methodologies. The media and incubation conditions were for total viable count (TVC) (CFU/g), plate count agar medium (tryptone glucose yeast agar) for 72 h at 45 ℃. Total coliform count (TCC) (CFU/g), chromogenic E. Coli/Coliform medium for24 h at 37 ℃. Yeast and moulds, The plates were incubated in a culture incubator.
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